SECTION 1

Background and Experimental Design

The purpose of this mini-report is to present one of the ways we can use a simple transcriptomic analysis to gain insight into possible biomarkers for disease presence or progression.

The analysis laid out here is not meant to readily translate into a clinical outcome without necessary follow-up studies and exhaustive review.

The dataset used in this presentation is based on an actual study: “Kim SK, Kim SY, Kim JH, Roh SA, Cho DH et al. (2014) A nineteen gene-based risk score classifier predicts prognosis of colorectal cancer patients”.

The dataset contains RNA-seq data from 18 subjects, but sampled (and this is very important) from different tissues of these subjects, normal large intestine tissue, primary colon cancer and progressive liver metastasis (colon originating). The final number of samples is actually 54 but for this brief report 37 relevant samples, 18 normal and 19 primary colon cancer samples will be used.

The basic logic for any potential bio-marker discovery is to compare the cancer samples to non-cancer samples and observe the differences in their gene expressions.

For any potential biomarker discovery the goal is to compare the cancer samples to non-cancer samples and observe the differences in their gene expressions.

Overview of data

Run Sample.Characteristic.biopsy.site. Sample.Characteristic.Ontology.Term.biopsy.site. Sample.Characteristic.disease. Sample.Characteristic.Ontology.Term.disease. Sample.Characteristic.disease.staging. Sample.Characteristic.Ontology.Term.disease.staging. Sample.Characteristic.individual. Sample.Characteristic.Ontology.Term.individual. Sample.Characteristic.organism. Sample.Characteristic.Ontology.Term.organism. Sample.Characteristic.organism.part. Sample.Characteristic.Ontology.Term.organism.part. Factor.Value.biopsy.site. Factor.Value.Ontology.Term.biopsy.site. Analysed
SRR975551 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_2 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975552 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_3 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975553 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_5 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975554 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_6 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975555 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_7 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975556 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_8 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975557 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_9 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975558 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_10 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975559 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_12 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975560 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_13 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975561 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_17 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975562 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_18 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975563 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_19 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975564 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_20 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975565 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_21 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975566 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_22 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975567 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_23 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975568 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_24 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colon http://purl.obolibrary.org/obo/UBERON_0001155 primary tumor http://www.ebi.ac.uk/efo/EFO_0000616 Yes
SRR975569 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_2 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975570 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_3 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975571 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_5 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975572 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_6 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975573 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_7 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975574 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_8 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975575 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_9 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975576 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_10 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975577 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_12 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975578 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_13 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975579 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_17 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975580 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_18 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975581 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_19 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975582 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_20 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975583 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_21 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975584 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_22 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975585 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_23 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975586 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_24 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975587 normal http://purl.obolibrary.org/obo/PATO_0000461 colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_2 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 colonic epithelium http://purl.obolibrary.org/obo/UBERON_0000397 normal http://purl.obolibrary.org/obo/PATO_0000461 Yes
SRR975588 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_3 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975589 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_5 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975590 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_6 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975591 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_7 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975592 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_8 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975593 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_9 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975594 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_10 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975595 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_12 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975596 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_13 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975597 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_17 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975598 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_18 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975599 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_19 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975600 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_20 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975601 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_21 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975602 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_22 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975603 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_23 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes
SRR975604 colorectal cancer metastatic in the liver colorectal cancer http://www.ebi.ac.uk/efo/EFO_0005842 Stage IV Colorectal Cancer NA AMC_24 NA Homo sapiens http://purl.obolibrary.org/obo/NCBITaxon_9606 liver http://purl.obolibrary.org/obo/UBERON_0002107 colorectal cancer metastatic in the liver Yes

As it can be seen the first 18 samples are primary tumors (colorectal cancer) and the next 19 are normal and then the rest are liver metastasis samples. For this tutorial we will use only the first 37, so primary tumor and normal samples.

Running DESEQ2

Creating DESeq results object using Benjamini-Hochberg correction

baseMean log2FoldChange lfcSE stat pvalue padj
ENSG00000000003 1006.252417 0.4171339 0.2540846 1.641713 0.1006496 0.2084175
ENSG00000000005 6.572053 -0.0476418 0.4225178 -0.112757 0.9102232 0.9473385
ENSG00000000419 370.323717 0.7674698 0.1742659 4.404016 0.0000106 0.0002247
ENSG00000000457 179.743933 -0.1868380 0.1148662 -1.626571 0.1038282 0.2134028
ENSG00000000460 58.131389 0.9189451 0.2353201 3.905086 0.0000942 0.0010838
ENSG00000000938 68.277653 0.4785311 0.2157950 2.217526 0.0265872 0.0766179

Out of 40166 non-zero gene counts there are 121 or 0.3% or upregulated genes and 30 or 0.075% of down regulated genes. So that would be around 151 genes of interest out of over 50 000. First conclusions is that there are much more upregulated genes of interest as potential causal factors for this type of cancer, but the 30 downregulated are not something to ignore either.

One thing is very important and that is the multiple testing correction procedure. Over 40 000 nonzero genes means that the analysis will include over 40 000 hypothesis tests. FWER or family wise error rates occurs at high rates in these situations, finding positive results by random is almost certain if multiple correction procedure is implemented. That’s why DESeq always has some multiple correction procedures.

I added the pAdjustMethod = ‘BH’, and this means using Benjamini-Hochberg correction, I did this on purpose so you can see how to implement the multiple testing procedures, even tough its implemented by default. But multiple testing procedure is very important in this case and I want a more robust method implemented. Therefore I will use the Holm multiple testing adjustment, which is going to be more robust on FWER.

Heatmap

The heatmap in this section is merely an overview of the data though it might not be as informative since we have a lot of genes in this dataset so it becomes really hard to track them. However this is good a snapshot of relative expression differnces

Volcano plot to display differentially expressed genes and their relative significance

Selecting only marker genes of interest using the adjusted p-value.

SECTION 2

Gene Set Functional Enrichment analysis

Summary

To explore analyze the data more closely, I also did a short trial run and extracted some gene sets from the Kyoto Encylopedia of Genes and Genomes (KEGG) for colorectal cancer to check if there was significant enrichment of those specific gene sets. The result from the KEG colorectal cancer gene set shows that the specific gene sets in that dataset are not significantly enriched in our dataset as the p-ad = 0.73 and the NES (normalized expression) is -0.0879.

GSEA RESULT AND PLOT

## # A tibble: 19,190 × 2
##    Gene       stat
##    <chr>     <dbl>
##  1 A1BG-AS1 -4.70 
##  2 A1CF     -1.56 
##  3 A2M      -0.474
##  4 A2M-AS1  -0.886
##  5 A2MP1    -1.80 
##  6 A4GALT    0.949
##  7 AAAS      1.21 
##  8 AACS     -0.332
##  9 AADAC     1.60 
## 10 AADACL2  -4.34 
## # ℹ 19,180 more rows

GSEA Plot

This plot is generated using the normalized enriched values for

Conclusions

Increased expression of CCNB1, CLCA1, and PLK4 restrained proliferation as well as migration of cancer cells and induced apoptosis of cancer cells. CCNB1, KIF4A, TPX2, MT1F, PRC1, PLK4, CALD1, MMP9, CLCA1, and MMP1 were identified as hub genes and CCNB1, CLCA1, and PLK4 could inhibit the progression of colon cancer. These highly expressed genes are also used as prognostic indicators in colorectal cancer.

The top 10 pathways that are significantly enriched based on this analysis include the typical cancer pathways that we expect to have genes that exhibit high expression. The supplementary study to this analysis was a survival analysis based on expression of the key colorectal cancer genes and the relevant pathway expression modules. It is important to note that this analysis report is a tip of the iceberg as there are many possible additional analyses that would need to be done in order to have a more granular view of the data that will in turn inform clinical decision making.

SECTION 3 : Potential Future directions

  1. Final interpretation would be that the interesting genes were identified starting with COL11A1, CEMIP, ADAM12, MMP1, OTOP3 (check volcano plot above in Section 2);

  2. Further experimental research would be needed to conclude if any of these are potential cancer treatment targets;

  3. Whole Exome Seq followed by variant curation to enahnce the insights we have obtained from this analysis;

  4. CCNB1, KIF4A, TPX2, MT1F, PRC1, PLK4, CALD1, MMP9, CLCA1, and MMP1 were identified as some of the genes and CCNB1, CLCA1, and PLK4 could inhibit the progression of colon cancer.